Universal collection medium

ABSTRACT

This invention provides a novel universal collection medium for cell collection. The medium allows for the first time the ability to perform cytology and direct molecular analysis on cells preserved in a single sample. This invention also provides novel methods for analyzing cells to assess human conditions.

This is a continuation of prior application Ser. No. 09/598,571, filedJun. 21, 2000 now abandoned, to which priority under 35 U.S.C. §120 isclaimed.

FIELD OF THE INVENTION

The present invention is generally related to the field of cytologicaland molecular assays and specifically to the area of assays for theassessment of conditions using cytological and molecular assays.

BACKGROUND OF THE INVENTION

The detection and diagnosis of human conditions is of obvious importancefor the treatment of disease. Numerous characteristics of diseases havebeen identified and many are used for their diagnosis. Many diseases arepreceded by, and are characterized by, changes in the state of theaffected cells. Changes can include the expression of viral genes ininfected cells, changes in the expression patterns of genes in affectedcells, and changes in cell morphology. The detection, diagnosis, andmonitoring of diseases can be aided by the assessment of such cellstates.

Routinely, for patients suspected of having one or more infectiousdiseases, for example human papilloma virus or herpes simplex virus, asample of cells is taken from the patient for analysis. Generally, sucha sample is in the form of a swipe or cellular scrape from the areaprimarily affected by the disease. These swipes usually collect amixture of normal and diseased cells with a very limited total number ofcells. The collected cells are traditionally smeared onto a slide forfurther analysis. When biochemical analysis was attempted, it was doneat the expense of a cytological analysis and was done via qualitativemethods such as in situ hybridization.

Routinely, the cervical sample obtained for conventional cytology issmeared onto a slide for morphological analysis. If this sampleidentifies potential disease by cell cytology, the patient must returnfor colposcopy to have a second sample collected for repeat cytologyand/or genetic analysis and other molecular tests such DNA, RNA orprotein. Recently, liquid cytology media have appeared on the market,which provide for enhanced morphology. These media were discovered to beamendable to molecular tests such as for HPV DNA, however, cells areroutinely collected into 10-20 ml of preserving agents, whichexcessively dilute DNA, RNA and other asssayable biomolecules, makingmolecular testing less than ideal. Further, while the current preservingreagents preserve cellular morphology these reagents allow degradationof DNA and RNA, such that quantitative analysis becomes difficult orimpossible upon storage.

Thus, present day analysis requires at least two samples to be obtainedfrom a patient in order to determine cell morphology and quantitativegenetic analysis. Current cytology methods use large volumes of apreserving agent which excessively dilute DNA, RNA and other assayablebiomolecules. Further, while the current preserving reagents preservecellular morphology these reagents allow degradation of individualbiomolecules such as DNA, RNA and protein, such that quantitativeanalysis becomes impossible.

Designing methods and media to preserve cells or tissue for analysis byboth morphological and biochemical analysis has proven problematic inthe past. For example, fixing cells or tissue for morphological analysiscompromised the ability to do biochemical analysis. Preserving cellularRNA was done at the expense of DNA and morphological analysis.Similarly, preserving cellular DNA was done at the expense of RNA andmorphological analysis.

It is an object of the present invention to provide a general collectionmedium for identifying cellular morphology and quantitatively preservingDNA, RNA and other biomolecules from a single sample regardless of thedisease to be detected.

It is another object of the present invention to provide a universalcell collection medium for preserving cell morphology and nucleic acids,lipids, carbohydrates, or proteins in a cell sample where limitednumbers of cells are available in the sample.

It is another object of the present invention to provide compositionsand methods for solution-based direct analysis of biomolecules ofinterest. The methods and compositions of this invention solve problemsencountered in non-solution-based methods such as in situ hybridizationor non-direct methods which require separation of the biomolecule ofinterest from other cellular components before analysis.

SUMMARY OF THE INVENTION

The present invention relates to the detection, analysis and monitoringof cellular disease. A new cell collection medium is disclosed whichpreserves both cell morphology and cellular biomolecules forquantitative analysis in a cell sample so that multiple assays can becarried out from a single patient sample. The state of the cells can beassessed using a device for collecting cellular samples in a smallvolume.

One embodiment of the present invention involves examining the cellmorphology and detecting a specific DNA sequence or measuring the levelsof expression of genes involved in a cell state, and comparing theirexpression to each other or to reference genes in a specific ratio, asan indication of the state of a disease in the cells. This method can beused to detect and/or monitor the onset or progression of any humancondition which causes a change in cell morphology or in levels orstructures of specific biomolecules. For example, the present inventioncan be used to assess predisposition to a particular disease or toassess the stage or risk of a disease as indicated by the state of thecells. It can also be used to guide or assess the effectiveness of atherapy for a disease by identifying appropriate therapy based on theindicated cell state or by indicating any change in the state of cellssubjected to the therapy.

In an other embodiment of the invention, a universal cell collectionmedium is disclosed. This medium allows simultaneous preservation ofcell morphology and biomolecules in a small volume. Also embodied in thepresent invention is a device for collecting such cell samples.

In a further embodiment of the invention, methods and compositions areprovided wherein a sample is analyzed according to cell morphology andbiochemical analysis in solution phase. The biochemical analysis iseither qualitative or quantitative and directly analyzes RNA, DNA,protein, carbohydrate or any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. UCM 127, baseline (12 hours at RT). CaSki cells.Magnification×200. Papanicolaou staining.

FIG. 2. UCM 127, 3 weeks at RT. CaSki cells. Magnification×200.Papanicolaou staining.

FIG. 3. UCM 127, 6 weeks at RT. CaSki cells. Magnification×200. H&Estaining.

FIG. 4. UCM 128, (12 hours at RT). CaSki cells. Magnification×200.Papanicolaou staining.

FIG. 5. UCM 128, 3 weeks at RT. CaSki cells. Magnification×200.Papanicolaou staining.

FIG. 6. UCM 128, 6 weeks at RT. CaSki cells. Magnification×400. H&Estaining

FIG. 7. UCM 130, baseline (12 hours at RT). CaSki cells.Magnification×200. H&E staining.

FIG. 8. UCM 130, 3 weeks at RT. CaSki cells. Magnification×200.Papanicolaou staining.

FIG. 9. UCM 130, 6 weeks at RT. CaSki cells. Magnification×400. H&Estaining.

FIG. 10. PreservCyt (134), baseline (12 hours at RT). CaSki cells.Magnification×200. H&E staining.

FIG. 11. PreservCyt (134), 3 weeks at RT. CaSki cells.Magnification×200. H&E staining.

FIG. 12. PreservCyt (134), 6 weeks at RT. CaSki cells.Magnification×400. H&E staining.

FIG. 13. CytoRich (135), baseline (12 hours at RT). CaSki cells.Magnification×200. Papanicolaou staining.

FIG. 14. CytoRich (135), 3 weeks at RT. CaSki cells. Magnification×200.Papanicolaou staining.

FIG. 15. CytoRich (135), 6 weeks at RT. CaSki cells. Magnification×400.H&E staining.

FIG. 16. UCM 141, 6 weeks at RT. CaSki cells. Magnification×400. H&Estaining.

FIG. 17. UCM 149, 6 weeks at RT. CaSki cells. Magnification×400. H&Estaining.

FIG. 18. UCM 141, baseline at RT. Normal epithelial and CaSki cells.Magnification×200.

FIG. 19. UCM 141, baseline at 4° C. Normal epithelial and CaSki cells.Magnification×200.

FIG. 20. UCM 141, 10 days at RT. Normal epithelial and CaSki cells.Magnification×200.

FIG. 21. UCM 141, 10 days at 4° C. Normal epithelial and CaSki cells.Magnification×200.

FIG. 22. UCM 141, 10 days at RT. Normal epithelial and CaSki cells.Magnification×200.

FIG. 23. UCM 141, 10 days at 4° C. Normal epithelial and CaSki cells.Magnification×200.

FIG. 24. UCM 149, baseline at RT. Normal epithelial and CaSki cells.Magnification×200.

FIG. 25. UCM 149, baseline at 4° C. Normal epithelial and CaSki cells.Magnification×200.

FIG. 26. UCM 149, 10 days at RT. Normal epithelial and CaSki cells.Magnification×200.

FIG. 27. UCM 149, 10 days at 4° C. Normal epithelial and CaSki cells.Magnification×200.

FIG. 28. UCM 149, 3 weeks at RT. Normal epithelial and CaSki cells.Magnification×200.

FIG. 29. UCM 149, 3 weeks at 4° C. Normal epithelial and CaSki cells.Magnification×200.

FIG. 30. PreservCyt, baseline at RT. Normal epithelial and CaSki cells.Magnification×200.

FIG. 31. PreservCyt, baseline at 4° C. Normal epithelial and CaSkicells. Magnification×200.

FIG. 32. PreservCyt, 10 days at RT. Normal epithelial and CaSki cells.Magnification×200.

FIG. 33. PreservCyt, 10 days at 4° C. Normal epithelial and CaSki cells.Magnification×200.

FIG. 34. PreservCyt, 3 weeks at RT. Normal epithelial and CaSki cells.Magnification×200.

FIG. 35. PreservCyt, 3 weeks at 4° C. Normal epithelial and CaSki cells.Magnification×200.

FIG. 36. CytoRich, baseline at RT. Normal epithelial and CaSki cells.Magnification×200.

FIG. 37. CytoRich, baseline at 4° C. Normal epithelial and CaSki cells.Magnification×200.

FIG. 38. CytoRich, 10 days at RT. Normal epithelial and CaSki cells.Magnification×200.

FIG. 39. CytoRich, 10 days at 4° C. Normal epithelial and CaSki cells.Magnification×200.

FIG. 40. CytoRich, 3 weeks at RT. Normal epithelial and CaSki cells.Magnification×200.

FIG. 41. CytoRich, 3 weeks at 4° C. Normal epithelial and CaSki cells.Magnification×200.

FIG. 42. Conventional Pap smear (archival). Magnification×120.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a universal cell collection medium thatmakes it possible to conveniently collect and preserve cells and theircontents for assessment of the existence or progression of a diseaseisolated from a single small patient sample, using cytological assays,molecular assays, or both.

The instant universal cell collection medium preserves cell morphologyand preserves macromolecules in a cell sample for either qualitative orquantitative analysis. One useful form of the disclosed cell collectionmedium preserves nucleic acids in the cells. Such preservation can belimited to refrigerated samples. Alternatively, preserved samples can bekept at ambient temperatures. Different forms of the universalcollection medium preserve a sample for days or weeks or more. Theuniversal cell collection medium can be used to collect cell samples forany purpose and is not limited to use with any particular assay method.Some forms of the universal collection medium contain a buffered salineisotonic solution or an alcoholic solution such as methanol, ethanol, ora similar alcohol, an RNase inhibitor such as RNasin, and a proteaseinhibitor such as pepstatin. Many different nuclease inhibitors areknown in the art, including, for example, vanadate complexes, chelatingagents and detergent-based compounds as well as specific inhibitors suchas RNasin. Any known nuclease and/or protease inhibitors can be employedin the present invention as a component of the universal collectionmedium in order to preserve the particular molecules of interest in thea sample.

The formulations of this invention provide for the first time means forperforming cytological and molecular analysis on cells which arecontained in a single sample. The cells are obtained from a patient andstored in the UCM of this invention. From this single sample, cells areextracted and a cytological examination is performed, the cellular DNAis qualitatively or quantitatively examined, the cellular RNA isqualitatively or quantitatively examined, or any combination of analysisis performed. The different analyses are performed concurrently or, forexample, after the results of the cytological analysis are obtained, thecells are subjected to molecular analysis days, weeks or even monthslater. Conversely, after molecular analysis of the cells contained inthe UCM, for example by automated screening, the sample is retrieved forcytological analysis, days, weeks or even months later.

In one embodiment of the invention the universal collection medium (UCM)formulations of this invention are buffered, water-based solutions whichcomprise a preservative such as a mixture of one or more alcohols, across-linking agent and an agent to inhibit degradation of RNA, DNA andprotein. The use of the UCM formulation is further enhanced by theaddition of an antimicrobial agent.

Nearly any non-viscous alcohol can be used to formulate the UCM, forexample, any C1 to C10 alcohols or mixtures thereof can be used.Preferred alcohols include methanol, ethanol, propanols, butanols, andpentanols. Most preferred are ethanol and n-butanol. The alcohol cancomprise a significant percentage of the formulation. For example, thealcohol(s) component can comprise about 1% to about 75% of the UCMformulation. More preferred is the percentage range of about 1% to about50% alcohol and more preferred is about 5% to about 30% alcohol in theUCM formulation. A range of about 5% to about 15% alcohol is alsopreferred.

The pH range of the UCM formulation is important for maintaining thecellular biochemical and morphological integrity of the cells. A pHrange of about 2.5 to about 6 is used to formulate the UCM of thisinvention. More preferred is a pH range of about 3 to about 5 and mostpreferred is a pH range of about 3.5 to about 4.5. Buffer(s) are used tomaintain the pH of the UCM at a constant value. Any buffer that hasbuffering capacity in the indicated pH range can be used in the UCM ofthis invention. Non-limiting examples of buffer components includeglycine, maleic, phosphoric, tartaric, citric, formic, or acetic acidsand the like.

The cross-linking agents of this invention comprise about 1% to about25% of the UCM formulation. Preferably, the cross-linking agentscomprise about 1% to about 15% or from about 1% to about 10%. Mostpreferably, the cross-linking agent comprises about 1% to about 5% ofthe UCM formulation. Cross-linking agents (also known as fixatives) arewell-known in the art (see, for example, Stedman's Medical Dictionary,25^(th) Edition, Williams & Wilkins, Baltimore Md. 1990 at page 592) andtheir use in the UCM formulation is now readily apparent to those ofskill in the art in light of the present invention. Non-limitingexamples of the cross-linking agents for use in the UCM formulationinclude aldehydes such formaldehyde, glutaraldehyde and the like. Apreferred cross-linking agent is glutaraldehyde-bisulfite.

Fixatives are defined by Stedman's as “serving to fix, bind, or makefirm or stable.” They are substances “used for the preservation of grossand histologic specimens of tissue, or individual cells, usually bydenaturing and precipitation or cross-linking the protein constituents.”Non-limiting examples of such fixatives for use according to the presentinvention are polymers such as poly(ethylene glycols) and poly(ethyleneoxides). Poly(ethylene glycols) are preferred and poly(ethylene glycols)(“PEG”) with a molecular weight of between about 600 and about 4,600 aremore preferred. PEG having a molecular weight of 1500 (“PEG-1500”) ismost preferred. Fixatives, which function in an equivalent manner tocross-linking agents for the purpose of this invention, are formulatedas described above for cross-linking agents.

Agents able to inhibit degradation of RNA, DNA and/or protein arewell-known in the art. They can work by either inhibiting enzymes orsequestering metal ions or both. Nuclease or protease inhibitors such asRNasin or pepstatin or chelating agents can be used according to thisinvention. Preferred agents to inhibit degradation of RNA, DNA and/orprotein are chelating agents. Chelating agents are well-known in the art(see, for example, Data For Biochemical Research, Third Ed., Rex M. C.Dawson et al., Oxford University Press 1986, at chapter 17) and areknown to both attenuate metal ion-induced and enzymatic degradation ofbiopolymers. Non-limiting examples of chelating agents for use in thisinvention include murexide, chromotropic acid,1-(1-hydroxy-2-napththylazo)-2-hydroxy-5-nitronaphthalene-4-sulphonicacid, EDTA (ethylenediaminetetraacetic acid), o-phenanthroline, thioureaand the like. A preferred chelating agent is EDTA.

Antimicrobial agents for use in this invention are those known in theart. Non-limiting examples of antimicrobial agents are aminoglycosides,β-lactams, cephalosporins, macrolides, penicillins, azides and the like.A preferred antimicrobial agent is sodium azide.

The universal collection medium can be used for a combination of two ormore assays of different characteristics related to a cell state ofinterest. As used herein, the assay or assays refer to detection ormeasurement of specific characteristics, the results of which may becombined with other such measurements of other characteristics toprovide an overall assessment of a cell suspected of being infected withone or more diseases. These assays may include, for example, acombination of morphological analysis and quanitation of a particularRNA or DNA or protein or carbohydrate structure whose presence or levelsprovide a specific indication of the presence or progression of adisease.

The universal collection medium can be used to collect any desired cellsample. Cell samples are collected in any suitable manner, includingscrapings, biopsy, or washings, and from any suitable source. Numerouscell collection techniques are known and any can be used with thepresent invention. Generally, the source of cells for a cell sample ischosen based on the known or likely tissue affected by the cell state ofinterest.

Cell samples for use in the present invention can be collected andstored in liquid medium. Examples of useful cell collection media arePreservCyt® (Cytyc), and CytoRich™ (Autocyte). These media weredeveloped for the collection of cytological samples but can be adaptedfor use with molecular assays when modified as described herein.

Nucleic acid detection generally benefits from the use of a reagentcapable of preventing nucleic acid degradation prior to performing theassay if the assay is not performed soon after sample collection. Auseful medium is a preservative based collection medium that hasstabilizers for nucleic acids (both RNA and DNA) and proteins and thatpreserves cell morphology, such as the universal collection medium ofthe present invention.

One method useful with the present invention involves measuring thelevels of expression of genes involved in a disease state, and comparingtheir expression to each other or to reference genes, as an indicationof the state of the cells. Such measurements can be combined with otherassays to increase the accuracy and reliability of the assessment of thedisease state. The present invention can be used to assess the stage ofa disease as indicated by the state of the cells. This embodiment canalso be used to guide or assess the effectiveness of a therapy for adisease by identifying appropriate therapy based on the indicateddisease state or by indicating any change in the state of cellssubjected to the therapy. Also disclosed is a cell collection medium forpreserving cell morphology and cellular biomolecules in a cell sample sothat multiple assays can be carried out on the same sample.

Many diseases and other human conditions are characterized by specificcellular phenotypes and gene expression patterns. Such diseases andconditions can be identified and/or monitored by assessment of specificcellular morphology or levels or structures of particular biomolecules.For example, neoplastic and cancerous cells generally exhibit certaindistinctive morphologies and growth characteristics. Molecularcharacteristics, such as gene mutations and gene expression patterns arealso a good indicator of disease progression. Virally infected cells canexhibit different morphologies and gene expression patterns, includingexpression of viral genes. Using the present invention, thecharacteristics of the cell state, such as changes in cell morphologyand/or expression of genes can be determined from a patient sample.

The characteristics to be detected are specific to the cell state ofinterest and the disease suspected of being present in the cell sample.Such characteristics can be generally divided into two types,cytological characteristics and molecular characteristics. As usedherein, cytological characteristics are characteristics such as, forexample, overall cell shape and appearance of the cell and itsorganelles. The primary identification and classification of manyneoplastic and cancerous cells has traditionally been accomplished usingcytological characteristics. Identification of cytologicalcharacteristics is generally slow, requires a relatively high level oftraining, and generally cannot be easily automated. As used herein,molecular characteristics are the presence and/or absence and state ofparticular molecular species, such as proteins, nucleic acids,carbohydrates and metabolites. Such molecular characteristics aregenerally identified by detecting and/or quantifying the particularmolecules of interest.

The present invention allows both cytological and molecularcharacteristics to be analyzed from a small patient sample. Thecharacteristics assayed can include additional or surrogate markercharacteristics that are not a direct cause or result of the disease butthat are related to certain disease and cell states. Examples of suchadditional markers include polymorphic markers, human leukocyte antigens(HLA) such as B7 that predispose women for cervical carcinomas,oncogenes, p53 mutations, BRCA1/2 mutations, other cancer markers,oncosuppressors, cytokines, growth factor receptors, and hormones. Suchmarkers can be present in, or absent from, cells exhibiting state- ordisease-specific characteristics, and such presence or absence can beindicative of, for example, a more severe or less severe disease state.These markers can be used in conjunction with disclosed methods to infereither higher or lower risk of neoplastic disease depending on thenumber of abnormal scores or the magnitude of change in quantitativemarkers.

Examples of disease states for assessment using the present inventioninclude, but are not limited to, autoimmune disorders, neoplasias, andcancer. Other disease states of interest include HPV-based diseaseincluding HPV infection, cervical intraepithelial neoplasia (CIN), andcancer, atypical squamous cells of undetermined significance (ASCUS),warts, epidermo dysplasia verruciformis and other skin diseases,laryngeal papilloma, oral papilloma, conjunctival papilloma and prostatedisease including enlarged prostate and prostate cancer, chlamydia, andviral infections such as HIV and herpes.

A cell sample as the term is used herein is primarily a collection ofcells from a patient. One method of obtaining cells is throughnon-invasive means, which is defined herein as obtained without thepuncturing of a patient. Examples of non-invasive means are, forexample, cell samples obtained from urine or a nasal, epithelial,cervical or other cell surface scrape. Other methods for obtaining acell sample are by needle biopsy, or tissue biopsy. The cells arecollected into volumes of less than 10 ml. More preferably, the cellsare collected into volumes of less than about 5 ml, and most preferredthe cells are collected into volumes of less than about 2 ml.

Combinations of multiple assays may be used with the media and device ofthe present invention and can be carried out from the collection and useof a single sample. An important aspect of the combination of assays isthe use of a universal cell collection medium that allows a single cellsample to be used for multiple assays of different types with a minimumnumber of assay-specific processing steps required. For example, cellsfor assaying cytological characteristics are typically collected in alarge volume of liquid which leaves the cell sample too dilute for mostassays of molecular characteristics. Prior art methods do not providefor direct methods for both cytological and molecular analysis. Theprior art methods require several extra steps, such as a separateconcentration step, which is inconvenient and may result in many of themolecular components of the cell being degraded. Cytological cellsamples, while optimized for preserving cell morphology, generally donot preserve nucleic acids well; thus making the sample unusable formany molecular assays.

Cell samples for use in the method of the present invention can be fixedor processed in any manner consistent with the assays to be performed.For example, both cytological and molecular assays can be performedusing cells fixed on a solid substrate such as a slide. Preferably,however, molecular assays are done in solution. The requirements of theassays to be performed will generally determine the sample processing tobe used.

The types of comparisons described above can also be used with manydifferent genes and disease states. That is, the measured level ofexpression of a gene of interest can be compared, for example, to thelevel of expression of the same type of gene in a different cell sample(such as an earlier cell sample from the same source or appropriatereference cells), to the level of expression of a different type of genein the same or a different cell sample, to the level of expression of areference gene in the same cell sample, or to the level of expression ofa reference gene in reference cells.

Expression of genes of interest can be assessed using any suitablemethod. For example, RNA can be detected using hybridization,amplification, or sequencing techniques, and protein, like carbohydrate,can be detected using specific antibodies. Many techniques for thespecific detection of gene expression, by detection of expressionproducts, are known and can be used with the disclosed UCM. Onetechnique for detecting and measuring the level of expression of genesof interest is detection of RNA transcribed from the genes of interest.For the most reliable comparisons, expression levels that are to becompared should be measured using the same technique and be performed inthe same manner.

Useful techniques for measuring the level of expression of a gene ofinterest in a cell sample include the hybrid capture technique describedin WO 93/10263 by Digene, PCR in situ hybridization techniques describedby (Nuovo, 1997)), branched DNA assays (Chernoff (1997)), transcriptionmediated amplification (TMA); Stoflet (1988)), and polymerase chainreaction (PCR), ligase chain reaction (LCR), self-sustained sequencereplication (3SR), nucleic acid sequence based amplification (NASBA),strand displacement amplification (SDA), and amplification with Qβreplicase (Birkenmeyer and Mushahwar, (1991); Landegren, (1993)).Numerous assays for the detection and measurement of gene expressionproducts are known and can be adapted for the determination of the levelof expression of genes of interest using the UCM-collected samples.

The present invention provides methods for analyzing cells thatpreviously were unavailable. For example, a combination of cancerous andnon-cancerous cells were placed in a Universal Collection Mediumaccording to this invention. The morphology of the cells was examinedafter storage at either 4° C. or room temperature for periods of 12hours, 10 days, 3 weeks and 6 weeks. Slides were prepared by stainingusing procedures well-known in the art, for example, Papanicolaoustaining and Hematoxylin and Eosin (H&E). When the slides were examinedunder a light microscope, all the features characteristic for cancercells were visible and well-preserved in all cases. The slides showedlarge and hyperchromatic nuclei, usually with oval shape and irregular,granular chromatin, scanty cytoplasm, multinucleated cells, presence ofnucleoli and presence of mitotic figures. In addition, the slides showedthat there was good cell dispersion and no cell clumping. Both types ofcells (normal and cancerous) had distinct and sharp shapes of both thenuclei and cytoplasm. The nuclei were well-stained and the normal cellsshowed a different color of cytoplasm depending on the maturity of thecell.

In one embodiment of the present invention RNA was analyzed directly bysolution based procedures. The cells were first lysed by adding aproteolytic enzyme to the cells contained in wells of a microtiterplate. Non-limiting examples of enzymes for use in the present inventioninclude proteinase K or Pronase. Cells can also be subjected todetergent lysis or osmotic lysis or a French Press. After incubation,biotinylated DNA probes were added to each well. The RNA:DNA hybridswere captured onto a solid phase by transferring to streptavidin coatedmicroplates. Alkaline phosphatase-conjugated antibodies to RNA:DNAhybrids were added to each well in the hybridization microplate andsignals were generated by adding a chemiluminescent reagent such asCDP-Star™ with Emerald II (Tropix) to each well. The signal was readfrom the microplate. The solution based DNA analysis was performedsimilarly to the RNA analysis except that the microtiter plates werecoated with anti-RNA:DNA hybrid antibodies and the probes were unlabeledRNA probes.

The present invention can be conveniently performed using kits thatinclude one or more of the materials needed for the method, such asreagents and sample collection and handling materials. For example, kitscan include cell collection medium including sample preserving reagents,reagents for specific detection of DNA sequences, RNA sequences and/orexpression products (mRNA or protein) of one or more DNA sequences, andsample handling containers. Useful reagents for detection of DNAsequences and/or RNA sequences are nucleic acid probes or proteinnucleic acid probes for those sequences. Useful reagents for detectionof DNA sequence expression products (proteins) are antibodies. Aberrantcarbohydrate antigens associated with cancerous cells can also bedetected by antibodies. A kit may also contain control samples orreagents, or reagents and materials for performing other assays to becombined with the disclosed assay.

The present invention can be performed using devices adapted to themethod. Numerous devices for performing similar assays are known and inuse and can be adapted for use with the disclosed UCM formulations,assays and methods. For example, devices are known for automating all ora part of sample assays and sample handling in assays.

All or part of the disclosed method can be controlled or managed usingspecial purpose computer programs. The data collected from the disclosedmethod, and data from any other assay used in combination, can becompiled, analyzed, and output in various forms and for various purposesusing special purpose computer programs. Such programs can be used with,or combined into, other patient or data management computer programs.The usefulness of such a program increases with the number ofmeasurements or assessments to be combined, and the relative importanceof each type of measurement to the overall assessment. Computer programsfor use with the disclosed method can be used on general purposecomputers, or can be incorporated into special purpose computers orcomputerized devices for controlling the disclosed method, handling andanalyzing data from the disclosed method, or both.

EXAMPLES

The examples herein are meant to exemplify the various aspects ofcarrying out the invention and are not intended to limit the inventionin any way.

Formulations

Formulation 127

-   -   20% Ethanol    -   0.05% NaN₃    -   5 mM EDTA    -   2.5% Glutaraldehyde-Sodium-Bisulfite (G-S-B)    -   0.2 M NaOAc-HOAc    -   pH 3.7        Formulation 128    -   20% Ethanol    -   0.05% NaN₃    -   5 mM EDTA    -   2.5% Glutaraldehyde-Sodium-Bisulfite (G-S-B)    -   0.2 M NaOAc-HOAc    -   pH 4.1        Formulation 130    -   7% Butanol    -   0.05% NaN₃    -   5 mM EDTA    -   2.5% Glutaraldehyde-Sodium-Bisulfite (G-S-B)    -   0.2 M NaOAc-HOAc    -   pH 3.7        Formulation 134    -   PreservCyt® (Cytyc Corporation)    -   Contains buffered methanol    -   Storage limits with cytologic samples: 3 weeks at 4° C.-37° C.        Formulation 135    -   CytoRich™ (AutoCyte Corporation)    -   Contains less than 24% alcohol    -   Storage 15° C.-30° C.        Formulation 141    -   20% Ethanol    -   0.05% NaN₃    -   5 mM EDTA    -   2.5% Glutaraldehyde-Sodium-Bisulfite (G-S-B)    -   0.2 M NaOAc-HOAc    -   pH 4.4        Formulation 149    -   10% Butanol    -   0.05% NaN₃    -   5 mM EDTA    -   2.5% Glutaraldehyde-Sodium-Bisulfite (G-S-B)    -   0.2 M NaOAc-HOAc    -   pH 4.4        Formulation 167    -   9% Butanol    -   0.05% NaN₃    -   5 mM EDTA    -   2% PEG-1500    -   10 mM NaOAc-HOAc    -   pH 4.5

Example 1 General Methods for Nucleic Acid Analysis

The assay for nucleic acids follows in general principle the method fordetecting HIV RNA by the Digene Hybrid Capture HIV Test, described in WO93/10263 by Digene. Briefly, following lysis, 50 μl of probe mix(containing DNA biotinylated probe) was added to each well. The platewas sealed and incubated at 65° C. for 1.5 hours for hybridization tooccur. After hybridization, samples were transferred to astrepavidin-coated microplate, and 25 μL of anti-hybrid antibody wasadded to each well. The plate was agitated at 1100 RPM, for 1 hour, atroom temperature. Wells were washed 6X times with 65° C. wash buffer,followed by one wash using distilled water. 100 μl of a chemiluminescentsubstrate was added to each well and the plate was incubated at roomtemperature for 30 minutes. The plate was then read in the DML 2000luminometer. The data was then expressed as signal-to-noise. Using acalibration curve, the chemiluminescent signal generated by eachspecimen was converted into mRNA copies per cell.

Example 2 General Methods for Morphological Analysis

HPV 16 positive cancer cells (CaSki) were placed in UCM 127, 128, 141,130, 149 and in two commercially available fixatives PreservCyt (CytycCorp.) and CytoRich (AutoCyte). The samples were then stored at ambienttemperature. The baseline slides were prepared after 12 hours ofstorage. Then slides were prepared after 3 and 6 weeks. In additionslides were prepared from UCM 141 and 149 after 6 weeks at RT. Theslides were prepared by spotting 200 μl of cell suspension ontopolycarbonate filter. The filter was then placed on the glass slide andblotted. The filter was then removed and the slides fixed in 95% ethanolfor 5 minutes. The slides were stained using routine Papanicolaoustaining and Hematoxylin and Eosin (H&E). The slides were evaluatedunder a light microscope using different magnifications, anddocumentation in the form of color pictures was prepared. Additionally asmaller study was performed using a mixture of normal human cervicalcells and CaSki cells. This mixture was placed in two UCM formulations141 and 149 and in PreservCyt and CytoRich controls. Each sample wassplit into two tubes and placed at RT and at 4° C. After 12 hours(baseline), 10 days and three weeks of storage, slides were prepared andstained with Papanicolaou staining. The following features wereevaluated for the morphology study: cell dispersion, cell shape, nuclearshape, chromatin pattern and staining intensity, nuclear/cytoplasmratio, presence of nucleoli, cytoplasm shape and staining color. FIGS.1-17 show the morphology of CaSki cells stored in different UCM and inPreservCyt and CytoRich controls stored at RT for 12 hours, 3 weeks and6 weeks. All features characteristic for CaSki cells (carcinoma cells)were visible and well preserved in all fixatives tested after 3 and 6weeks: large and hyperchromatic nuclei usually with oval shape andirregular granular chromatin, scanty cytoplasm, multinucleated cells,presence of nucleoli and presence of mitotic figures. In addition, theslides showed that there was good cell dispersion and no cell clumpingin all media tested.

FIGS. 18-41 show the morphology of normal human cervical cells mixedwith CaSki cells and stored in UCM 141, 149 and PreservCyt and CytoRichcontrols for 12 hours, 10 days and 3 weeks at room temperature and at 4°C. The evaluation of these slides showed that the morphology was wellpreserved in UCM and in PreservCyt and CytoRich controls. After 3 weeksat 4° C. and room temperature both types of cells (normal cervical andCaSki cells) had distinct and sharp shapes (both nuclei and cytoplasm).The nuclei were well stained and the normal cells showed a differentcolor of cytoplasm depending on the maturity.

FIG. 42 was taken from an archived routine Pap smear to show themorphology and staining of samples stored fixed to slides forcomparison.

Example 3 HC II HPV DNA Assay Results

UCM formulations 127, 128, 130 and the STM™ (Digene) control were testedusing the Hybrid Capture II HPV DNA Test. A standard HC II HPV Test kit(Digene catalog number 5101-1096) was used. Each collection medium (1mL) was spiked with 0.8×10⁶ CaSki cells (˜500 copies/cell). Thisconcentration of CaSki cells was chosen because an adequate clinicalspecimen usually contains about 1×10⁶ cells. The same stock of CaSkicells was then used for morphology study and DNA and RNA testing. Astandard volume of 50 μl was used per assay as described in the PackageInsert, without any sample preparation modification. A similar STMsample was prepared by spiking the same number of CaSki cells into 1 mLof Digene Sample Transport Medium (STM—this medium is the current mediumused for HPV testing. It preserves DNA and RNA but not cell morphology).The HC II HPV test was performed at day “0” (baseline) and after one andsix weeks of storage at room temperature. Table 1 shows the resultsobtained for each medium at the baseline and after one and six weeksexpressed as S/N ratios.

UCM formulations 127 and 128 had the highest S/N ratios comparable tothe STM control S/N ratios at the baseline and retained these valuesafter storage at RT for six weeks. The S/N ratios for UM 130 obtained atthe baseline were slightly lower when compared to UCM 127 and 128. UCM130 retained its signal after 6 weeks.

TABLE 1 Signal to Noise ratios obtained for UCM 127, 128, 130 and STM atbaseline and after one week and six weeks storage at RT. Week 1 Week 6Baseline % of original % of original Medium S/N S/N signal S/N signal127 5666 5852 103 6298 111 128 5760 5669 98 5932 103 130 4407 5135 1175500 125 STM 5557 6151 110 6127 110

Formulations UCM 141 and 149, used in the smaller morphology study, weretested in the HC II HPV DNA assay at baseline and then after one, twoand four weeks of storage at room temperature. CaSki cells (1.5×10⁶)were spiked in these media and 50 μl were used per assay. Table 2 showsthe S/N ratios. Both formulations retained close to 100% (UCM 141 98.4%and UCM 149 97.3%) of the original signal after four weeks of storage atroom temperature.

TABLE 2 S/N ratios obtained for formulations UC 141 and 149 at baselineand after 1, 2 and 4 weeks of storage at RT. Week 1 Week 2 Week 4Baseline % original % original % original UCM S/N S/N signal S/N signalS/N signal 141 10875 11006 101.2 10427 95.9 10703 98.4 149 9443 815986.4 8035 85.3 9188 97.3

In addition, samples stored in PreservCyt and CytoRich were examinedaccording to the protocols of this example. Both the PreservCyt andCytoRich media were found to be deficient in their ability to provideDNA for satisfactory analysis.

Example 4 HC II RNA Assay Results

The aliquots of all formulations (CaSki cells 0.8×10⁶) were stored at 4°C. instead of room temperature and were tested in the HC II HPV RNAassay. The prototype RNA assay used in this study utilized biotinylatedsingle-stranded DNA probes specific for HPV 16 E6/E7 RNA. UCMformulations 127, 128 and 130 were tested at baseline and after one andsix weeks storage at 4° C. UCM formulations 141 and 149 were tested atbaseline and after one, two and four weeks of storage at 4° C. Tables 3and 4 show the results.

TABLE 3 Results obtained for UCM 127, 128 and 130 in HC II RNA assay atbaseline and after one and six weeks of storage at 4° C. Week 1 Week 6Baseline % original % original Medium S/N S/N signal S/N signal 127 9371157 123 990 106 128 981 1139 116 1192 122 130 845 1261 149 1278 151

All UCM formulations tested had comparable S/N ratios when tested at thebaseline. UCM 127, 128 and 130 retained a 100% signal after six weeks ofstorage at 4° C. in comparison to baseline results. RNA in cells in bothUCM formulations 141 and 149 appears to be stable at 4° C. for fourweeks giving S/N ratios close to the baseline results (UCM 141 108% andUCM 149 89%).

TABLE 4 Results obtained for UCM 141 and 149 in HC II RNA assay atbaseline and after one, two and four weeks of storage at 4° C. Week 1Week 2 Week 4 Baseline % original % original % original UCM S/N S/Nsignal S/N signal S/N signal 141 1074.5 1029 96 1137 106 1164 108 1491243.5 1786 144 1843 148 1103 89

In addition, samples stored in PreservCyt and CytoRich were examinedaccording to the protocols of this example. Both the PreservCyt andCytoRich media were found to be deficient in their ability to provideRNA for satisfactory analysis.

Publications cited herein and the material for which they are cited arespecifically incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

REFERENCES

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1. A cell collection medium, wherein the cells contained in the mediumcan be analyzed directly by both cytological and molecular methods,wherein the molecular method of analysis comprises either RNA or DNA orprotein analysis or the analysis of both RNA and DNA, wherein the mediumis water based and comprises a buffer component, at least one alcohol, anon-crosslinking fixative and an agent to inhibit degradation of atleast one of RNA, DNA, or protein, wherein the non-crosslinking fixativecomprises about 1% to about 15% of the medium, and wherein the buffercomponent has a buffering capacity within a pH range of about 3 to about5.
 2. The medium of claim 1, wherein the buffer component has abuffering capacity within a pH range of about 3.5 to about 4.5.
 3. Themedium of claim 1, wherein the alcohol component comprises a C1 to C10alcohol.
 4. The medium of claim 3, wherein the alcohol component isselected from the group consisting of methanol, ethanol, propanols,butanols, and pentanols.
 5. The medium of claim 4, wherein the alcoholcomponent comprises ethanol or n-butanol.
 6. The medium of claim 1,wherein the fixative comprises a polymer selected from the groupconsisting of a poly(ethylene glycol) and a poly(ethylene oxide).
 7. Themedium of claim 1, wherein the fixative is a poly(ethylene glycol). 8.The medium of claim 7, wherein the poly(ethylene glycol) has a molecularweight of between about 600 and about
 4600. 9. The medium of claim 8,wherein the poly(ethylene glycol) has a molecular weight of about 1500.10. The medium of claim 1, wherein the agent to inhibit degradation ofat least one of the group consisting of RNA, DNA, and protein comprisesat least one agent selected from the group consisting of a nucleaseinhibitor, a protease inhibitor and a chelating agent.
 11. The medium ofclaim 10, wherein the agent to inhibit degradation of at least one ofthe group consisting of RNA, DNA, and protein comprises a chelatingagent.
 12. The medium of claim 11, wherein the chelating agent isselected from the group consisting of murexide, chromotropic acid,1-(1-hydroxy-2-napththylazo-2-hydroxy-5-nitronaphthalene-4-sulphonicacid, ethylenediaminetetraacetic acid, o-phenanthroline, and thiourea.13. The medium of claim 12, wherein the chelating agent comprisesethylenediaminetetraacetic acid.
 14. The medium of claim 1, wherein thealcohol is selected from the group consisting of methanol, ethanol,propanols, butanols, and pentanols, wherein the agent to inhibitdegradation is a chelating agent selected from the group consisting ofmurexide, chromotropic acid,1-(1-hydroxy-2-napththylazo-2-hydroxy-5-nitronaphthalene-4-sulphonicacid, ethylenediaminetetraacetic acid, o-phenanthroline, and thiourea,and wherein the fixative comprises a polymer selected from the groupconsisting of a poly(ethylene glycol) and a poly(ethylene oxide).